High quality, continuous throughput, tissue fixation-dehydration-fat removal-impregnation method

ABSTRACT

A process and apparatus for rapid, continuous flow histological processing of tissues is disclosed. The steps of fixation, dehydration, clearing and impregnation are performed in less than one hour; this allows a pathologist to evaluate samples shortly after receipt, perhaps while the patient is still in the operating room. Rapid and continuous processing is accomplished by decreasing the thickness of tissue sections, use of non-aqueous solutions composed of admixtures of solutions, solution exchange at elevated temperature and with agitation, and impregnation under vacuum pressure. The patient in surgery is thus provided with point-of-care surgical pathology.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appln. Ser. No.60/056,102, filed Aug. 20, 1997, the entire disclosure of which isincorporated herein by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the rapid, continuous flow, processingof tissue for microscopic examination, from fixation to impregnation.

2. Description of the Related Art

Conventional methods prepare tissues for histology by incubation inseparate solutions of phosphate-buffered 10% formaldehyde for fixation,a series of increasing concentrations of ethanol for dehydration, andxylene for clearing tissue of dehydration agent, prior to impregnation.Because of the time required for this process, usually 8 hours orlonger, it is customary to complete these separate steps—fixation,dehydration, clearing, and impregnation—overnight in automatedmechanical instruments designed for those tasks (see, for example, U.S.Pat. Nos. 3,892,197, 4,141,312, and 5,049,510). A typical automatedtissue processor (TISSUE-TEK) requires more than eight hours and isprogrammed to process batches of tissue samples as follows.

Set Time Set Volume of Station Solution Concentration (min) TemperatureP/V** Agitation Solution 1 Buffered 10% 50 40° C. On On 2.2-3.2 Formalinliters 2 Buffered 10% 50 40° C. On On 2.2-3.2 Formalin liters 3 Alcohol*80% 50 40° C. On On 2.2-3.2 L 4 Alcohol 95% 50 40° C. On On 2.2-3.2 L 5Alcohol 95% 50 40° C. On On 2.2-3.2 L 6 Alcohol 100%  50 40° C. On On2.2-3.2 L 7 Alcohol 100%  50 40° C. On On 2.2-3.2 L 8 Alcohol 100%  5040° C. On On 2.2-3.2 L 9 Xylene 100%  50 40° C. On On 2.2-3.2 L 10 Xylene 100%  50 40° C. On On 2.2-3.2 L 11  Paraffin 50 60° C. On On 412  Paraffin 50 60° C. On On 4 13  Paraffin 50 60° C. On On 4 14 Paraffin 50 60° C. On On 4 **pressure/vacuum cycle *the alcohol used inmost laboratories is a mixture of 90% ethyl, 5% methyl and 5% isopropylalcohol.

Such conventional methodology demands that the tissue specimens be sentfrom the operating room, medical office or other sites, to a pathologylaboratory on one day; the tissue specimens be prepared overnight; andthe pathologist render a diagnosis based on microscopic examination oftissue sections the next day at the earliest, almost 24 hours afterdelivery of the specimen to the laboratory (FIG. 1). In addition to theminimum one-day delay in giving a surgeon the benefit of a report fromthe pathologist, there are also problems associated with impeded workflow in the pathology laboratory necessitated by the requisite batchprocessing of specimens, the safety concerns that attended havinginstruments operating overnight, the risk of possible instrumentfailures and the need to monitor the instruments, and the waste of usinglarge volumes of reagents for such processing when automated. Moreover,expensive measures are required to prevent exposure of laboratorypersonnel to fumes and toxic substances associated with the reagentsused in this process. Also, the large volumes of solvent waste andparaffin debris produced by conventional methodology pollute theenvironment.

Conventional fixation and processing cause irreversible damage to thestructure of DNA and particularly RNA that limits the application ofgenetic techniques for diagnosis and research. Consequently, most DNAand certainly RNA analysis require special precautions with handling ofmaterial, such as immediate freezing of fresh tissues, becauseretrospective genetic analysis is impaired by conventional tissueprocessing techniques.

Histological diagnosis of a frozen section suffers from multipledisadvantages in comparison to sections prepared from paraffin blocks:the slide prepared from a frozen section “does not possess . . .uniformity of quality”; “it is technically more difficult for serialsections of the same specimen to be examined”; “extreme caution must beexercised in cutting the specimen in order to ensure a sufficiently thinsection and to avoid the possibility of damaging details of thespecimen”; and all the slides must be prepared “while the tissue is inthe initial frozen state” because, “[i]f the tissue is thawed andrefrozen for sectioning, it is severely damaged” (U.S. Pat. No.3,961,097).

There is an ever present interest in expediting tissue processing andanalysis for diagnostic purposes. Furthermore, recent healthcare focushas been directed to lessening the cost of various procedures includingtissue processing. The costs of tissue processing are related to time,the space required for preparation and analysis, reagents (both theamount required for processing and handling discard), and the number ofpersonnel required. More importantly, patients and their physiciansdepend on evaluation and diagnosis by the pathologist to guidetreatment. Reducing the amount of time needed to complete tissueprocessing would lessen the anxiety experienced during the periodbetween obtaining the specimen and delivering the pathologist's reportto the surgeon.

Others have recognized the need to shorten the time required for tissueprocessing, but they have made only modest improvements in theconventional methods. To accelerate tissue processing, U.S. Pat. Nos.4,656,047, 4,839,194, and 5,244,787 use microwave energy; U.S. Pat. Nos.3,961,097 and 5,089,288 use ultrasonic energy; and U.S. Pat. No.5,023,187 uses infrared energy. U.S. Pat. No. 5,104,640 disclosed anon-aqueous composition of a fixative, a stabilizing agent, and asolubilizing agent that adheres a blood smear to a slide. However, theaforementioned patents do not teach or suggest that the entire processof preparing diagnostic tissue slides could be accomplished in less thantwo hours, starting from fixation and ending with impregnation, withcontinuous throughput of samples. The present invention provides such aprocess.

SUMMARY OF THE INVENTION

It is an object of the invention to provide compositions for tissueprocessing and an apparatus and system for utilizing the same thatreduces the time required for tissue processing and analysis, andreduces the cost thereof by reducing time, the size of the laboratoryfacility, the volumes of reagents used, and the number of personnelrequired. This allows conversion of existing practice to rapid responsesurgical pathology for the patient undergoing an operation, and may evenallow point-of-care diagnosis by the pathologist in the vicinity of theoperating room.

With regard to the processing and analysis of solid tissue, a tissueslice must be on the order of 4 to 6 microns to be examined under amicroscope, whereas the thinnest slice of fresh tissue that can beobtained by cutting is about 1 mm with the typical slice being on theorder of 3 mm. In order to produce a sufficiently thin slice frommicroscopic examination, it is necessary to harden the tissue so that afiner slice can be obtained, e.g., by sectioning with a microtome. Thepresent invention greatly accelerates the tissue hardening process andthus turns the conventional overnight processing into a process whichtotals on the order of 40 minutes. Thus, we have developed a simple,safe, low cost, expeditious, and reliable method that permitspreparation of impregnated tissue blocks suitable for microtomesectioning in less than two hours from the moment tissue is received inthe pathology laboratory. This method allows continuous flow ofspecimens, is adaptable to automation, precludes the need for formalinand xylene with their noxious fumes, allows standardization of tissueprocessing, and requires considerably smaller volumes of reagents thanconventional methods. Either fresh or previously fixed tissues can beprocessed by the present invention.

In addition to the reduction in time required for tissue processing, therapid preparation of tissue by the present invention is capable ofpreserving tissue structures and morphology that were lost withconventional methods.

Moreover, studies with tissues processed with the invention disclosedherein indicate better preservation of DNA and particularly RNAextraction than with conventional processing methods. Thus, tissuesobtained in hospitals and other settings can be processed for bothhistologic and genetic studies soon after delivery to the laboratory,and archival material may be made available for future research andother applications. Improvements may be expected in the yield of geneticmaterial, the stability of the genetic material in archival form, thesize and integrity of the genetic material, and reducing chemicalmodification of the genetic material in comparison to the prior art.

An object of the invention is to provide a method and an apparatus forrapid processing of tissue for histology with continuous throughput. By“continuous throughput,” we mean accessing the system with additionalsamples, minutes apart. Therefore, at any given time there are samplesof tissue in different stages of processing. In other words, with ourmethod, there is continuous throughput and flow of specimens along thevarious steps of tissue processing. In contrast with our method, batchprocessing is presently required because conventional methodology takeseight hours or longer. Samples are placed in automated instruments,which can not be access with additional samples until the entireinstrument cycle is completed. All these tissue samples are at the samestage of processing at any given step of the instrument cycle.

Yet another object of the invention is to provide non-aqueous reagentsfor rapid, continuous flow processing of tissue for histology.

A further object of the invention is to eliminate the need for toxicsubstances such as formalin and xylene in tissue processing.

In accordance with one aspect of the invention, a tissue specimen isfixed, dehydrated, and fat is removed. A suitable admixture for use is anon-aqueous solution comprised of fixative and dehydrating agents,preferably a ketone and an alcohol; the volume ratio of alcohol toketone may be between about 1:1 to about 3:1. The tissue specimen isincubated for about 25 minutes or less, more preferably for about 15minutes or less, and even more preferably for about 5 minutes or less.Incubation is preferably between about 30° C. and 65° C., morepreferably between about 40° C. and 55° C., and most preferably betweenabout 45° C. and 50° C.

Another aspect of the invention is fixation, dehydration, fat removal,and clearing of a tissue specimen. A preferred solution in this aspectof the invention is alcohol and a clearant. This process may beaccomplished in about 5 minutes or less.

In yet another aspect of the invention, a tissue specimen is cleared andimpregnated in a single solution comprised of a clearant and animpregnating agent. Preferably, this process may be accomplished inabout 5 minutes or less. Prior to sectioning, the impregnated tissuespecimen may be embedded in the impregnating agent.

A tissue specimen which has been fixed, dehydrated, and defatted maythen be impregnated in a wax solution. Consistent with dehydration ofthe tissue specimen, the wax solution is preferably as low as possiblein water content. Thus, the wax solution may be prepared prior toimpregnation by heating the wax to evaporate any dissolved water and bydegassing under reduced pressure. Impregnation of the tissue specimenmay take place under less than atmospheric pressure and at elevatedtemperature to remove any solvents from the tissue specimen and to drawthe wax solution into the tissue specimen. Vacuum decreases impregnationtime by accelerating diffusion and reducing the evaporation temperatureof any solvents that may be present in the sample. The wax solution maycomprise degassed paraffin and/or mineral oil. Impregnation of thetissue specimen may be completed in about 15 minutes or less;preferably, completed in about 10 minutes or less. Prior to sectioning,the impregnated tissue specimen may be embedded in the impregnatingagent to form a tissue block.

Another embodiment of the invention is processing a tissue specimen fromfixation to impregnation in a series of solutions, at least some ofwhich are admixtures that perform more than one task at the same time:fixation, dehydration, removal of fat, and impregnation. The admixturemay include a fixative, a dehydrating agent, and a fat solvent (e.g.,ketone and alcohol). Another solution may include fixative, dehydratingagent, fat solvent, and clearant (e.g., alcohol and xylene). Yet anothersolution may include a clearant and an impregnating agent (e.g., xyleneand paraffin). The tissue specimen may be impregnated in a wax solutioncomprised of a mixture of different chain lengths (e.g., at roomtemperature, mineral oil which is liquid and paraffin which is solid).Preferably, an admixture contains at least two different chemicals(e.g., two alcohols).

Processing time may be reduced by a non-aqueous admixture (e.g.,fixative-dehydrating agent-fat solvent, fixative-dehydrating agent-fatsolvent-clearant, clearant-impregnating agent), microwave energy as asource to achieve uniform heating within the tissue specimen, andreducing the pressure by using a vacuum source. Diffusion of thesolution into the tissue specimen and chemical exchange may be promotedby mechanical agitation, heat, reduced pressure, or a combinationthereof.

The above steps may be accelerated by adding a fixative enhancer, asurfactant, or both to the solutions used in the process. The fixativeenhancer may be polyethylene glycol (PEG), mono- and dimethyleneglycol,propylene glycol, polyvinyl pyrrolidone, or the like; the polymer usedmay be between about 100 and about 500 average molecular weight,preferably about 300 molecular weight. The surfactant may be dimethylsulfoxide (DMSO), polyoxyethylene sorbitan esters (e.g., TWEEN 80),sodium dimethyl sulfosuccinate, mild household detergents, or the like.

The fixative may be a ketone (e.g., acetone, methyl ethyl ketone),aldehyde (e.g., acetylaldehyde, formaldehyde, glutaraldehyde, glyoxal),alcohol (e.g., methanol, ethanol, isopropanol), acetic acid, leadacetates and citrate, mercuric salts, chromic acid and its salts, picricacid, osmium tetroxide, or the like.

The tissue specimen may be dehydrated with methyl alcohol, isopropylalcohol, ethyl alcohol, propyl alcohol, butanol, isobutanol, ethylbutanol, dioxane, ethylene glycol, acetone, amyl alcohol, or the like.

Fat may be removed from the tissue specimen with an organic solvent suchas, for example, acetone, chloroform or xylene.

The clearant may be xylene, limonene, benzene, toluene, chloroform,petroleum ether, carbon bisulfide, carbon tetrachloride, dioxane, cloveoil, cedar oil, or the like.

The tissue specimen may be impregnated and/or embedded in paraffin,mineral oil, non-water soluble waxes, celloidin, polyethylene glycols,polyvinyl alcohol, agar, gelatin, nitrocelluloses, methacrylate resins,epoxy resins, other plastic media, or the like.

In the context of the invention, a “tissue specimen” is a piece oftissue that may be processed by the methods disclosed herein. It mayalso refer to single cells from any biological fluid (e.g., ascites,blood, pleural exudate), or cell suspensions obtained from aspiration ofsolid organs or lavage of body cavities. Single cells may be pelleted bysedimentation or buoyant centrifugation prior to processing.

The methods of the invention are specially suitable for tissue specimensin which cell—cell contact, tissue organization, organ structure, or acombination thereof must be preserved. Such a specimen is a tissue slicepreferably about 3 mm or less in its smallest dimension, more preferablyabout 2 mm or less, even more preferably about 1.5 mm or less, and mostpreferably about 1 mm or less.

The tissue specimen may be fresh, partially fixed (e.g., fixation in 10%formalin for 2-3 hours), or fixed (e.g., overnight fixation in 10%formalin or any other fixative). The above invention allows processingof a tissue specimen from fixation to impregnation in less than abouttwo hours, preferably less than about 90 minutes, more preferably lessthan about one hour, even more preferably less than about 45 minutes,and most preferably less than about 30 minutes. If the tissue specimenis fixed or partially fixed, then the processing time may be shortenedaccordingly. Tissue may be transported from the operating room to thepathology laboratory in an aqueous solution; such a transport solutionmay consist of equal volumes of an aqueous buffer and the non-aqueousadmixture described herein.

Following impregnation, the tissue specimen can be embedded to produce ablock. The agent used to embed the tissue specimen is preferably thesame as the material used for impregnation, but a different impregnatingagent may also be used. The blocked tissue specimen can be mounted on amicrotome to produce tissue sections of between about 1 micron and about50 microns, preferably between about 2 microns and about 10 microns. Thetissue sections may be further processed for histochemical staining,antibody binding, in situ nucleic acid hybridization/amplification, or acombination thereof. The tissue specimens are then typically examined bymicroscopy, but other techniques for detecting cellular properties maybe used to examine the processed tissue specimen (e.g., automatedcytometry, autoradiography, electrophoresis of nucleic acid).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing that almost 24 hours elapse between thetime a tissue specimen is obtained by a surgeon and the time a diagnosisby a pathologist can be prepared from microscopic examination ofsections of the tissue;

FIG. 2 is a flow chart showing that with the present invention,diagnosis by the pathologist can be made available to the surgeon whoprovided the tissue specimen in about 2 hours or less;

FIG. 3 is a schematic plan view of a tissue processing facility providedin accordance with the invention;

FIG. 4 shows an exemplary shaker bath provided as a part of theapparatus and system of the invention;

FIG. 5 shows an exemplary microwave oven for use as a part of theapparatus and system of the invention;

FIG. 6 shows an exemplary paraffin bath provided as a part of theapparatus and system of the invention;

FIG. 7 is a schematic illustration of a microwave/impregnation unitprovided in accordance with an alternative embodiment of the invention;

FIG. 8 is a schematic illustration of a slicing guide provided inaccordance with an exemplary embodiment of the invention;

FIG. 9 is a broken away view of a tissue clamp and slicing assemblyprovided in accordance with another embodiment of the invention;

FIG. 10 is a schematic illustration of a tissue holder provided inaccordance with a further embodiment of the invention;

FIG. 11 is a schematic illustration of a tissue cassette provided inaccordance with the invention for receiving small tissue samples;

FIG. 12 shows a tissue receiving and transporting jar with tissuecassette in accordance with an embodiment of the invention; and

FIGS. 13A-13B show agarose gel electrophoresis of DNA and RNA,respectively, prepared from processed tissue specimens. In FIG. 13A,lane 1 contains molecular weight standards, lane 2 contains a dilutedsample from a tissue specimen processed according to the presentinvention, lanes 3-4 contain DNA samples from tissue specimens processedaccording to the present invention, and lanes 5-6 contain DNA samplesfrom tissue specimens processed according to a conventional method. InFIG. 13B: lanes 1, 4 and 6 are blanks, lanes 2-3 are samples from tissuespecimens processed according to a conventional method, lane 5 containsan RNA sample from a tissue specimen processed according to the presentinvention, and lane 7 contains control RNA.

DETAILED DESCRIPTION OF THE INVENTION

A process and apparatus for rapid, continuous histological processing oftissues is disclosed. The steps of fixation, dehydration, fat removal,and impregnation can be performed in less than about two hours; thisallows a pathologist to evaluate samples shortly after receipt, perhapswhile the patient is still in the operating or recovery room. Patientanxiety can be reduced by reducing the time required for pathologicaldiagnosis. Rapid and continuous processing is accomplished by decreasingthe thickness of tissue specimens, use of non-aqueous solutions composedof admixtures, solution exchange at elevated temperature and withagitation, uniform heating of tissues and solutions with microwaveradiation, impregnation under vacuum pressure, or a combination thereof.

Fixation, dehydration, and removal of fat are required for thepreparation of tissue prior to impregnation. These steps are facilitatedby trimming the tissue to a suitable size prior to processing, and usingcassettes which hold such tissue blocks and allow their easy transferbetween solutions for fixation, dehydration, removing fat, andimpregnation.

Fixation initiates hardening of the tissue specimen, and may preservecell morphology by cross linking proteins and halting cellulardegradation. Without chemical fixation, endogenous enzymes willcatabolize and lyse the cell, and the tissue microanatomy will bealtered. Such fixatives may be a ketone, aldehyde, alcohol, acetic acid,heavy metals, chromic acid, picric acid, or osmium tetroxide.Indications that fixation was inadequate can include: disassociation oftissue structures, bubbles in tissue sections, poor and irregularstaining, shrunken cells, clumping of cytoplasm, condensation and lessdistinct nuclear chromatin, and autolysis/hemolysis of erythrocytes.

Dehydration removes water from the tissue specimen to promote hardening.Replacement of water in the tissue specimen with a dehydrating agentalso facilitates subsequent replacement of the dehydrating agent withmaterial used for impregnation. This solution exchange is enhanced byusing a volatile solvent for dehydration. The dehydrating agent may below molecular weight alcohols, ketones, dioxane, alkylene glycols, orpolyalkylene glycols. Failure to dehydrate the specimen can lead toinadequate impregnation, poor ribbon formation during sectioning, cleftsin tissue sections, dissociation of structures, water crystals in tissuesections, and poor staining.

Fat in the tissue specimen is removed with a solvent because fat impairsclearing and impregnation. Inadequate fat removal can result inspreading artifacts of tissue sections, wrinkling of tissue sections,and poor staining.

Optionally, the tissue specimen is cleared. The clearant extractsdehydrating agent from the tissue specimen and reduces its opacity.Examples of clearants include xylene, limonene, benzene, toluene,chloroform, petroleum ether, carbon bisulfide, carbon tetrachloride,dioxane, clove oil, or cedar oil.

Finally, once the tissue specimen is suitably fixed and dehydrated, itis hardened by impregnation with an agent such as wax, celloidin,polyalkylene glycols, polyvinyl alcohols, agar, gelatin,nitrocelluloses, methacrylate resins, epoxy resins, or other plastics.Appropriate hardening of the tissue specimen with adequate preservationof cellular morphology is required prior to placing the impregnatedspecimen in a block and obtaining ten micron or thinner sections with amicrotome knife. Preferred impregnation materials are commercial waxformulae, mixtures of waxes of different melting points (e.g., liquidmineral oil and solid paraffin), paraplast, bioloid, embedol, plasticsand the like. Paraffin has been chosen for use in the examples hereinbecause it is inexpensive, easy to handle, and ribbon sectioning isfacilitated by the coherence of structures provided by this material.

If processing of the tissue specimen is incomplete, the sections cut bythe microtome knife will appear cracked or “exploded”. Tissue processingis deemed a failure when one or more of the following problems isencountered: embedded tissue blocks are too soft or too hard, sectionsfall out or show an amount of compression different from the embeddingagent, sections appear mushy, tissue ribbons fail to form or arecrooked, sections crumble or tear, erythrocytes are lysed, or clumpingof cytoplasm, condensation of chromatin, basophilic staining ofnucleoli, shrunken cells, spreading artifacts and moth-eaten effect.

For wax-impregnated sections on glass slides, the wax may be melted andremoved prior to staining or immunohistochernistry. The tissue sectionis rehydrated and then analyzed as described below with stains orantibodies. After staining is completed or the histochemical reaction isdeveloped, the slide may be coverslipped and viewed under a microscope.Alternatively, the stained or antibody-decorated specimen may be studiedwith an instrument for cytometry. The tissue blocks may be stored forarchival purposes or retrospective studies.

The present invention is compatible with preparation of nucleic acids,DNA or RNA, from processed tissues. Thus, genetic study is possible forspecimens collected routinely in the clinical pathology laboratory. Thecombined power of these technologies will be great. Histologicalobservations may be correlated with genetics by analyzing one section bystaining or immunohistochemistry, and preparing nucleic acids from anadjacent section for genetic analysis. For example, diseased and normalregions of the same section may be compared to detect geneticdifferences (e.g., mutations, levels of transcription), diseaseprogression may be characterized by comparing genetics differences insamples taken at several time points, and tumor evolution may beassessed by following the accumulation of genetic differences fromprimary cancer to metastasis.

Many features distinguish the present invention: (a) thin slicing of thetissues prior to processing; (b) continuous input of tissue specimens,and continuous flow through the system; (c) elimination of water fromsolutions (i.e., non-aqueous solutions); (d) fixation, dehydration, fatremoval, clearing, and impregnation of tissue performed with uniformheating (e.g., microwave energy); (e) admixture solutions tofix-dehydrate-remove fat, fix-dehydrate-remove fat-clear, andclear-impregnate; and (f) impregnation of tissue under reduced pressurewith degassed impregnating agent. These features make the presentinvention simple, practical, easy to implement, and amenable toautomation.

Hematoxylin-eosin staining is commonly used for histological study andmay be considered a standard for comparison by pathologists. Inaddition, the present invention has been found to be compatible withother stains including trichrome, reticulin, mucicarmine, and elasticstains as described in general references such as Thompson (SelectedHistochemical and Histopathological Methods, C. C. Thomas, Springfield,Ill., 1966), Sheehan and Hrapchak (Theory and Practice ofHistotechnology, C. V. Mosby, St. Louis, Mo., 1973), and Bancroft andStevens (Theory and Practice of Histological Techniques, ChurchillLivingstone, New York, N.Y., 1982). Such staining procedures would takebetween 30 minutes and several hours to complete, although rapidstaining procedures are available from Fisher Scientific that requireonly five minutes to accomplish.

Tissue may be obtained from an autopsy, a biopsy (e.g., endoscopicbiopsy), or from surgery. For cancer surgery, the ability to provide apathological diagnosis from a stained tissue section will provide thesurgeon with information that may be used prior to the patient'sdeparture from the operating room. For example, an indication from thepathologist that the cancer is confined to the resected tissue may allowthe surgeon to be conservative in treatment and to preserve neighboringhealthy tissue. Alternatively, a finding by the pathologist that canceris not confined to a resected organ would permit more aggressivesurgical treatment while the patient was still in the operating room.

Over 20,000 samples of tissue have been successfully processed by thepresent invention, including: brain, breast, carcinoma (e.g., bowel,nasopharynx, breast, lung, stomach), cartilage, heart, kidney, liver,lymphoma, meningioma, placenta, prostate, thymus, tonsil, umbilicalcord, and uterus. Mineralized tissue (e.g., bone, teeth) would requiredecalcification prior to processing by the present invention. Forexample, tissue may be decalcified with a hydrochloricacid/ethylenediaminetetraacetic acid (EDTA) solution from StephensScientific (Allegiance Healthcare Supply, catalog no. 1209-1A) accordingto the manufacturer's instructions.

Tissue sections processed by the present invention may also be used inimmunohistochemistry. The present invention provides tissue specimens inwhich antigen is recovered and preserved, the choice of fixative may beoptimized for recovery and preservation of particular antigens.Non-specific binding sites are blocked, antigen is bound by specificantibody (i.e., the primary antibody), and non-bound antibody isremoved. If labeled with a probe or signal generating moiety, theprimary antibody may be detected directly but it is preferred to attachthe probe to a protein (e.g., a secondary antibody) that specificallybinds the primary antibody. Secondary antibody may be raised against theheavy or light chain constant region of the primary antibody. Thisamplifies the signal generated by an antigen-antibody conjugate becauseeach primary antibody will bind many secondary antibodies.Alternatively, amplification may occur through other specificinteractions such as biotin-streptavidin. Antibody binding is performedin a small volume to reduce usage of expensive reagents and maintain ahigh binding rate; evaporation of this small volume is reduced byincubation in a humidity chamber. The signal generating moiety ispreferably an enzyme which is not otherwise present in the tissue. Forexample, alkaline phosphatase and horseradish peroxidase may be attachedto the secondary antibody or conjugated to streptavidin. Substrates areavailable for these enzymes that generate a chromogenic, fluorescent, orluminescent product that can be detected visually.

The staining pattern for antigen may be used to localize expression ofthe antigen in the context of cellular structures revealed bycounterstaining. Antigen expression can identify cell or tissue type,developmental stage, tumor prognostic markers, degenerative metabolicprocesses, or infection by a pathogen.

Antigen-antibody binding may also be visualized with radioactive,fluorescence, or colloidal metal probes by autoradiography,epifluorescent microscopy, or electron microscopy, respectively. Similarprobes may be used to detect nucleic acid in the tissue section by insitu hybridization to identify genetic mutations or transcripts;alternatively, the nucleic acid (DNA or RNA) may be extracted fromtissue sections and analyzed directly by blotting, or amplified prior tofurther genetic analysis.

Mutations may be germline and used to trace genetic predisposition ofdisease, or mutations may be somatic and used to determine geneticalterations in disease pathogenesis. The disease may be a metabolic orneurologic disorder, malignancy, developmental defect, or caused by aninfectious agent. The present invention preserves material for geneticanalysis by a simple procedure and room temperature storage.

It is envisioned that the present invention will preserve tissue thatyield greater amounts of nucleic acid with a higher average molecularweight than tissues processed by conventional processes.

In accordance with an exemplary system for tissue processing provided inaccordance with the present invention, a series of tissue processingstations may be provided, e.g., in a single tissue processing unit orarea. By way of non-limiting example, a suitable tissue processingfacility is illustrated in FIG. 3.

The first step in the process, which may be carried out at the tissueprocessing facility or elsewhere, is to prepare a suitable tissue samplefor hardening and ultimate examination. Typically, a slice of the tissueof interest is prepared. The finest slice possible is obtained, of about1 to 3 mm and preferably 1 to 2 mm in thickness. Processing time isproportional to the size of the tissue sample being processed. Thetissue slice is placed in a tissue cassette in which the tissue iscontained during the immediately following processing steps. The tissuecassette is next placed in a first solution provided in accordance withthe present invention.

By way of example, the cassette 10 may be placed in a conventionalbeaker 12, having the first solution 14 therein, preferably by itself asthe process described is a substantially continuous one, or togetherwith a limited number of other, similar tissue cassettes. The beaker 12is then placed in a shaker bath 16, as illustrated in FIG. 4, for gentlyagitating and heating the same. We have used a LAB-LINE/DUBNOFFincubator-shaker bath for this purpose. Rather than water, as it is ourgoal to minimize moisture to which the tissue samples are exposed and,in fact, ultimately to dehydrate the same, we have provided glycerine asthe temperature conducting fluid 18 in the shaker bath 16. Glycerine hasthe advantage that it is an effective conductor of thermal energy but itdoes not evaporate. Evaporation would undesirably increase the moistureof the environment in which the tissue is processed, and would requireperiodic replenishment. Because the glycerine neither needs replacementnor adds moisture to the environment, it is most preferred. For thisstage of the process, the tissue sample (in cassette 10) is disposed inthe first solution, in the shaker bath 18 for approximately 3-15minutes.

Supplemental agitation is desirably also provided during the shaker-bathstep. Presently, an external pump (A) (FIG. 3) is provided with a tube(not shown) therefrom inserted into the solution beaker 12 or otherreceptacle for bubbling and thus agitating its contents. An aerationdiffusion nozzle or plate may be provided to provide for more uniformsolution agitation as deemed necessary or desirable.

To ensure that the tissue cassette 10 and first solution containingbeakers 12 remain upright and in a desired disposition, we have modifiedthe conventional shaker-bath to provide transverse wires or stays 20,e.g., four wires, defining, e.g., five longitudinal channels in whichtissue cassette containing beakers 12 may be disposed. Thus, forexample, sample containing beakers 12 may be regularly added to theshaker-bath 18 and sufficiently processed tissue samples removed in turntherefrom for further processing as described hereinbelow, by adding newsamples on the left end of the shaker bath and removing sufficientlyprocessed samples from the right end thereof.

Next the tissue sample cassette 10 is exposed to a series of fluidswhile simultaneously being agitated and subjected to microwaveradiation. In the currently proposed embodiment, three microwave unitsare provided, as shown in FIG. 3, each having a different solution inwhich the tissue sample containing cassette is submerged for aprescribed period. In the alternative, a single source of microwaveenergy could be provided. However, such would require sequentialplacement of the respective solutions for receiving the tissue cassette.While for a single tissue sample such solution placement and replacementwould not significantly increase the duration of the tissue processingcycle, it can be appreciated that the use of a single microwave thatreceives multiple solutions, may hinder the continuity of the processwith respect to subsequent samples. Indeed, where a series of microwaveunits are provided, as a given tissue sample is moved from one microwaveto the next having the next solution, a subsequent tissue sample canthen be received in the first microwave unit. Thus, providing a unit foreach of the respective solutions means that a subsequent tissue sampleneed not be held while all microwave processing steps of the proceedingsample have been completed. It is to be understood, however, that withthe noted hindrance of continuity, the three microwave units illustratedcould be reduced to two or even one. Likewise, other steps in theprocess may be combined or sub-combined as deemed necessary or desirablefrom a balance of process continuity versus a potential reduction inmanpower, equipment, space requirements, etc. An exemplary such morecompact unit is discussed in greater detail below, with reference toFIG. 7.

With reference now to FIG. 5, an exemplary microwave unit 22 for tissueprocessing is illustrated. For applying microwave radiation, we arecurrently using laboratory microwave ovens obtained from Energy BeamSciences, Inc. We have used two microwave processor models, H-2800 andH-2500. Either model or another, similar such system could be used. Byway of example, a Pyrex or other clear microwaveable fluid receptacle 24is utilized to hold respectively second, third and fourth solutionsprovided in accordance with the invention in each of the three microwaveunits (FIG. 3). A temperature probe 26 is placed in the solution toensure that the temperature of the respective bath is within the desiredrange. Moreover, to provide for agitation, which accelerates the tissueprocessing, aeration is provided. The microwave units we have usedinclude a tube 28 for aeration. A single tube may be inserted into thebath, but for more uniform and complete agitation, it is most preferredto provide a diffusion plate or nozzle head (not shown in detail) incooperation with the gas tube 28 for diffusing the agitating bubbles,e.g., across a substantial portion of the diameter of the solutionreceptacle for uniform agitation of the entire volume of solution. Suchdiffusion plates and nozzles are well known and can be provided, e.g.,at the base of the solution receptacle.

Conventionally, paraffin is degassed as a part of the tissue processingprocedure. Degassing removes organic solvents from the paraffin. Toenhance this process, and to reuse the paraffin in the system we proposecontinuous degassing. This is accomplished by maintaining the vacuumwithin the covered Pyrex 32 at 640 mm. Hg.

Following the three sequential steps employing microwave radiation, thetissue sample cassette(s) are placed in a paraffin bath, as shown inFIG. 6. Currently, we provide a paraffin bath comprising three paraffinbath stations (beakers) 30 provided within a covered Pyrex jar 32. Forthe purpose of temperature control, the Pyrex jar 32 is placed in, e.g.,a Poly Science brand water bath 34. By applying a grease or the like tothe internal edges of the flanges on both the lid and jar, an airtightcoupling can be provided between the lid and jar and thus a vacuum canbe pulled through a tooled hose connector 36 provided in the lid.Suitable such Pyrex brand jars are available from Fisher Scientific. Wehave used Model No.01-092-25. To create a vacuum within the Pyrex jar32, a conventional pressure/vacuum pump 38 is coupled to a tube 40 thatis in turn coupled to connector 36. A suitable such power operated pumpis available from Fisher Scientific and has for example a 100 psi max.Agitation is preferably provided during the paraffin bath step, eitherthrough vibratory agitation, ultrasound, or potentially via aeration.

Next the tissue sample must be embedded. For that purpose we use aconventional Tissue-Tek embedding console system (I) (FIG. 3) availablefrom Miles/Sakura, e.g. Model No. 4708.

The embedded tissue sample is then cut in a conventional manner with amicrotome (L) (FIG. 3) and floated (M) for placement, we use the Leitz1512 Microtome, and the Lipshaw Electric Tissue Float Model 375.

After the slice is disposed on the slide, the slide is heated to removethe paraffin. We have used the Isotemp Oven 300 series available fromFisher (K) (FIG. 3).

Next the slides are stained. To accelerate the staining process, wepropose to use an automated stainer (O) (FIG. 3) to reduce the number ofpersonnel and time required. A non-continuous process could use theSakura diversified stainer DRS-601 which stains slides in batches;alternatively, a continuous process could use a Leica auto stainer XLwhich contains a dewaxing stage so that separate incubation in an ovenmay be omitted. The fixed and stained tissue sample is then covered,e.g. with the Tissue-Tek coverslipper, Manufacturer No. 4764 (R) (FIG.3).

As described above, the system for carrying out the dehydration andimpregnation in accordance with the invention can be a series ofdiscrete units. In the alternative, as also noted above, one or moresteps can be carried out in a single processing component or unit. Asalso discussed above, the number of units provided and the steps carriedout by each unit impacts the continuity of the processing unit. Thus, inlow volume environments, a single unit for carrying out a plurality ofthe tissue processing steps may be advantageous and will notsignificantly impact continuity of tissue processing. In higher volumesystems environments, two or more units may be preferred.

An exemplary combined unit 42 is illustrated in FIG. 7. The combinedunit 42 in fact includes two subunits; a microwave processor unit 44 andan impregnator unit 46. The microwave processor unit 44 is provided forsequentially submerging the tissue being processed in solution A,solution B, and solution C, in each instance agitating the solution andexposing the tissue to microwave energy. Thus, in the illustratedembodiment, a vessel 48 is provided for receiving for example one ormore trays 50 on which one or more tissue cassettes 10 may be placed.The vessel 48 is fluidly coupled to a source of each of the solutionsfor tissue dehydration. Thus, once the tissue cassette(s) are placed onthe respective tray(s) 50, solution A is conducted to the vessel 48 andmicrowave energy is applied thereto simultaneous to agitation via, forexample, an aeration tube (not shown in FIG. 7). After a sufficient timeof exposure has passed, solution A is drained and the tissue cassettesare preferably flushed either with solution B or with a combination ofsolution A and solution B so as to substantially eliminate residualsolution A. Solution B is then fed to the vessel 48 whereupon microwaveenergy and agitation are again applied for a prescribed period. At theconclusion of administration of solution B, solution B is returned to astorage vessel therefor and the tissue samples are flushed either withsolution C or a combination of solution B and solution C. Thereafter,solution C is fed to the vessel 48, agitation and microwave energy areapplied, and ultimately solution C is drained. The tissue samples arethen ready for impregnation.

In the illustrated embodiment impregnation is carried out in a secondsubunit 46 of the assembly. This allows impregnation to be carried outwhile a subsequent tissue sample(s) are subject to microwave energyapplication. If a single unit is provided, then the vessel used formicrowave processing can be used for impregnation however the microwaveenergy would not be applied thereto during the impregnation steps.

In accordance with the proposed impregnation process, a series ofparaffin solutions, e.g., 3 or 4, are applied to the tissue cassettesdisposed e.g. on suitable trays 52 in a vessel 54, to provide sequentialparaffin baths to effect the impregnation of the tissue sample as afinal step in the tissue preparation process. In the impregnator subunit46, the tissue samples are placed under a vacuum at a controlledelevated temperature. The tissue samples are preferably also agitatedduring this step with a magnetic stirrer, ultrasound, or air bubbler.

The remaining embedding, etc. steps of slide preparation are carried asoutlined above with reference to FIG. 3.

In accordance with the invention, additional, specialized instrumentsand apparatus have been developed to facilitate tissue processing ingeneral and in accordance with the invention, in particular. Thesespecially designed instruments and apparatus are described herein below.

As noted above, it is difficult to cut a thin slice of a solid tissuesample. On the other hand it is desirable, in terms of minimizingdehydration and fixation time, to have the tissue sliced as thinly aspossible in advance of the dehydration process. To facilitate creationof a thin slice we have proposed three instruments to aid thepathologist. One, for convenience referred to herein as a slicing guide60, as illustrated in FIG. 8, is in the form of a thin metal plate 62 onthe order of, e.g., 1 to 2 mm in thickness, having a cutout 64 the widthof, for example, a thumb nail (about 1 cm²). A stop 66 is defined at theend of the cutout or notch 64 to serve as a knife or blade stop. Tofacilitate picking up the slicing guide 60 from a flat surface or othercutting surface, a lip 68 may be provided at the end of the metal plate62, remote from the cutting notch. To provide a thin slice of tissue, alarger segment of tissue is placed over the cutout or notch 64 so that aportion thereof is disposed in the notch. Pressure is then applied tothe exposed surface of the tissue and a cutting instrument is placedagainst and slid horizontally along the slicing guide plate so as tosever the tissue disposed in the notch 64 from the remainder of thetissue. Engagement of the cutting blade with the blade stop 66 completesthe cutting process and the bulk of the tissue, disposed above the cut,is placed aside. The remaining tissue, disposed in the slot, can then beplaced in a suitable tissue cassette for dehydration and impregnation.

As can be appreciated, the slicing guide 60 facilitates the productionof a thin slice of tissue of generally uniform thickness which may befurther processed.

As another alternative for producing a thin tissue slice, we haveproposed to provide flat plates or blocks 70 at the end of an otherwiseconventional forceps 72, as schematically illustrated in FIG. 9. Theblocks may be permanently or temporarily secured to the ends of theforceps. This provides rather large, flat clamping surfaces 74. Thetissue to be cut may be placed between the clamping blocks 70 and asharp blade passed between the clamping blocks to slice the tissue. Bycutting closely to one of the two generally planar flat surfaces 74, athin tissue slice of generally uniform thickness can be provided. Theparallel rather large flat surfaces provide uniform pressuredistribution thus holding the tissue in position during the cuttingprocess and then ensuring a uniform cut that preferably preserves theintegrity of the tissue.

To hold the tissue in position during cutting we have also proposed athree prong fork-like instrument 92, illustrated in FIG. 10. In theillustrated embodiment the prongs 94 are spaced from each other byapproximately one centimeter and each has a sharp, pointed tip 96 tofacilitate penetration of the tissue with minimal disruption. By holdingthe tissue to a cutting board with the prongs 94 of the instrument 92,suitable slices of tissue can be obtained by cutting parallel to orbetween the prongs. In the illustrated embodiment, the instrument 92 ischaracterized in that the prongs have a length on the order of 5-10centimeters to accommodate a variety of specimens and a handle of about8 centimeters in length, itself spaced from the prongs by 2-4centimeters, to facilitate manipulation of the instrument and a suregrip during cutting. We have found that the fork-like instrument 92 isparticularly advantageous in obtaining sections from organs such as theintestine and gallbladder. Indeed, securing such specimens with prongs94 prevents the various layers of tissue from sliding upon each otherduring the cutting process.

We have also proposed to provide a tissue receiving unit and cassettefor use in the operating room, to facilitate transport of tissue,particularly very small segments of tissue, for example those obtainedby needle biopsy. When such biopsied tissue is put directly into, forexample, a jar of suitable solution, it can often be difficult for thelab technician to retrieve the minute tissue sample from the jar and inparticular to ensure that all biopsied tissue is retrieved. Thus, asillustrated in FIGS. 11 and 12, we have proposed to provide tissuecassettes 10′ to the operating room for immediately receiving suchminute tissue samples.

To contain such tissue samples within the tissue cassette 10′, we haveprovided thin sheets of biopsy sponge material 80, which is an open cellplastic foam, at least one of which has a partial depth recess 82defined therein to provide, together with the other biopsy sponge acompartment for receiving the biopsied tissue. Thus, in the operatingroom the biopsied tissue can be disposed immediately in the recessedportion 82 of one of the biopsy sponges 80 and the tissue cassette 10′closed. To maintain the integrity of the tissue for transport to theprocessing lab, the tissue cassette 10′ is placed within ajar ofsuitable solution. To facilitate retrieval of the cassette and to ensurethat it is maintained fully submerged in the solution, we have provideda specimen jar 86 having a columnar support 88 projecting from the lid90 and having structure at the tip thereof 90 for coupling tocomplementary structure 84 on the tissue cassette 10′. FIG. 12 shows thetissue cassette 10′ attached by its top surface. However, alternativeattachment points are possible such as the bottom surface or the hingedside of the cassette. Furthermore, two or more cassettes may be attachedto the columnar support 88.

Thus the tissue cassette 10′ with the biopsied tissue therewithin can betemporarily secured to the distal end of the columnar support 88 andinserted into a suitable solution for transport. At the tissueprocessing lab, the lid 90 is removed from the jar 86 and the tissuecassette 10′ removed from the column 88. Any suitable fasteners such asvelcro type fasteners, plastic snap lock, dove tail slide connectors orother cooperative engagement structure can be provided to attach thetissue cassette 10′ to the support column 88. The solution within thespecimen jar 86 may be a transport (aqueous) solution or the first(non-aqueous) solution. It would be convenient to provide the specimenjar in the operating room with the cassette attached to the outside ofthe jar and then to invert the lid so that the cassette is immersed inthe solution within the jar after tissue is placed within the cassette.

The present invention will have many advantages over conventionalmethods in the areas of the practice of pathology, patient care,biomedical research, and education.

The availability of microscopic diagnosis of tissue samples within about40 minutes to about 2 hours after receipt will allow rapid, or evenreal-time, clinical interaction between surgical intervention andpathological evaluation. This may bring about significant improvementsin patient care by eliminating or reducing to a minimum patient anxietyduring the wait for diagnosis of disease, prognosis, and planning fortreatment.

Consequently, there will be a drastic reordering of the workflow inpathology laboratories. Clinical laboratory space, pathologicalexpertise, and clerical and technical personnel will be utilized moreefficiently. Continuous workflow will improve accessibility andresponsiveness of pathologists who process and evaluate specimens,reduce the number of pathologists needed to process and evaluatespecimens, and may also improve medical education, particularly theaccessibility and responsiveness of residency programs.

The smaller volume of reagents will result in cost savings. Eliminationof formaldehyde and xylene and the diminished requirement for otherhazardous chemicals will provide benefits to the environment andincreased safety in the laboratory.

Standardization of tissue fixation and processing procedures will easecomparison of specimens from different laboratories. Artifacts inhistology due to the use of formaldehyde and/or prolonged processingwill be eliminated; thus, allowing more precise evaluation ofmicroscopic morphology of normal and diseased tissues. Similarly,antigen retrieval and staining will be improved. For genetic analysis,formaldehyde-induced DNA mutations will be eliminated and extraction ofnucleic acid from archival material may be enhanced. The feasibility ofRNA studies from stored, fixed paraffin-embedded tissue opens unlimitedavenues for diagnostic and research applications.

All books, articles, applications, and patents cited in thisspecification are incorporated herein by reference in their entirety.

The following examples are meant to be illustrative of the presentinvention; however, the practice of the invention is not limited orrestricted in any way by them.

EXAMPLES Example 1

Two mm thick or thinner slices of fresh or previously fixed tissue wereheld in tissue cassettes and placed in a non-aqueous first solution of:

40% isopropyl alcohol,

40% acetone,

20% polyethylene glycol (average molecular weight 300), and

1% dimethyl sulfoxide (DMSO) (i.e., 10 ml per liter of the abovemixture).

Tissues samples were incubated for 15 min at a glycerin bath temperaturebetween 45° C. and 50° C. The 400 ml solution for fixation was placed ina 500 ml beaker in a water bath shaker (linear displacement of 5cm/sec). Additional agitation of the fixation solution was provided bybubbling with an air pump.

Fixation, dehydration, fat removal, clearing, and impregnation areaccomplished by sequential exposure of the tissue specimen to threedifferent solutions (the second, third and fourth solutions describedabove), one in each of three microwave ovens from Energy Beam Sciences.A one liter solution of 70% isopropyl alcohol and 30% polyethyleneglycol (average molecular weight 300) is placed in the first oven (modelH2800) in a 1500 ml beaker, the solution in the second oven (modelH2800) consists of one liter of 70% isopropyl alcohol and 30% xylene ina 1500 ml beaker, and the third oven (model H2500) contains a solutionof 1000 ml of xylene and 300 gm of paraffin in a 1500 ml beaker. Ten mlof DMSO per liter are added to these three solutions. Heating at 60° C.by microwave radiation is effected for 15 minutes in the first oven, and5 minutes each in the second and third ovens (75% power setting with acycle of 2 seconds).

To continue paraffin impregnation after completion of the microwaveradiation steps, tissue sections were incubated in four 500 ml baths ofmolten paraffin placed within a large dessicator filled with paraffin,and resting in a glycerin bath at 75° C. Tissue sections weretransferred from one paraffin bath to the next at 3 minute intervals,for a total impregnation time of 12 minutes. Each 3 minute interval wasmeasured from the time that the pressure reading is about 640 mm. Of Hg.No agitation was used during this step.

Example 2

Fixation, dehydration, fat removal, and paraffin impregnation of freshor fixed tissue sections, approximately 1 mm thick, was accomplished in40 minutes by exposing these tissue sections to four successive steps asfollows.

Step 1.

In this example, the first solution consisted of:

60% isopropyl alcohol,

10% acetone,

30% polyethylene glycol (average molecular weight 300), and

dimethyl sulfoxide (DMSO) added at an approximate concentration of 1% ofthe total volume. One liter of this solution suffices to fix 60 samplesof tissue held in tissue cassettes. The samples were incubated at 55° C.in a commercial tissue microwave processor (H2500 or H2800, Energy BeamSciences) for 5 min each in a series of three baths containing the firstsolution (15 min total incubation); agitation of the solution wasobtained by bubbling to accelerate solution exchange.

Step 2.

The samples were incubated in a solution of 70% isopropyl alcohol, 30%acetone, and DMSO added at an approximate concentration of 1% at 60° C.Samples were heated in a commercial tissue microwave processor (H2800,Energy Beam Sciences) for 5 min each in two beakers containing thesolution (10 min total incubation), which were agitated by bubbling.

Step 3.

Following microwave irradiation, impregnation was initiated byincubation in a wax solution of 25% mineral oil and 75% molten paraffinplaced in a large dessicator resting in a 60° C. or 70° C. glycerinbath, under a vacuum of about 200 mm of Hg, for 5 min. Paraffin wasdegassed prior to use as described in Example 1.

Step 4.

Impregnation was completed by incubation in four baths of moltenparaffin placed within a large dessicator resting in a glycerin bath at75° C. Tissue sections were transferred from one paraffin bath to thenext at 3 min intervals, for a total impregnation time of 12 min. Each 3min interval was measured for the time that the pressure reading isabout 640 mm of Hg.

In this example, 6 ml of a color indicator stock solution (10 μmmethylene blue in 1000 ml of isopropyl alcohol) was added to each of thesolutions of isopropyl alcohol and acetone. Tissue specimens acquire ablue tint that facilitates their handling during impregnation andhandling; penetration of the tissue specimen may also be monitored byobservation of an even blue color throughout the tissue specimen.

Example 3

Fixation, dehydration, fat removal, and paraffin impregnation of freshor fixed tissue sections, up to about 1 to 2 mm thick, may beaccomplished in 65 minutes as follows.

Step 1.

In this example, the first solution consists of:

40% isopropyl alcohol,

40% acetone,

20% polyethylene glycol (average molecular weight 300),

glacial acetic acid added at an approximate concentration of 0.5% of thetotal volume, and

dimethyl sulfoxide (DMSO) added at an approximate concentration of 1% ofthe total volume. One liter of this solution suffices to fix 60 samplesof tissue held in tissue cassettes. The samples are incubated at 65° C.in a commercial tissue microwave processor (H2500 or H2800, Energy BeamSciences) for 15 min in a 1500 ml beaker containing the first solution;agitation of the solution is obtained by bubbling to accelerate solutionexchange.

Step 2.

The samples are incubated in a solution of 55% isopropyl alcohol, 25%acetone, 10% polyethylene glycol (average molecular weight 300), 10% lowviscosity mineral oil, glacial acetic acid added at an approximateconcentration of 0.5% of the total volume, and DMSO added at anapproximate concentration of 1%. Samples are heated at 65° C. in acommercial tissue microwave processor (H2800, Energy Beam Sciences) for15 min in a 1500 ml beaker containing the solution, which is agitated bybubbling.

Step 3.

The samples are incubated in a solution of 55% isopropylic alcohol, 25%acetone 20% low viscosity mineral oil, glacial acetic acid added at anapproximate concentration of 0.5% of the total volume and DMSO added atan approximate concentration of 1% of the total volume. Samples areheated at 65° C. in a commercial tissue microwave processor (H2800,Energy Beam Sciences ) for 5 minutes in a 1500 ml beaker containing thesolution, which is agitated by bubbling.

Step 4.

Following microwave irradiation, impregnation is initiated by incubationin two baths of a wax solution of 30% low viscosity mineral oil and 70%molten paraffin placed in a large dessicator resting in a 60° C.glycerin bath, under a vacuum of about 640 mm of Hg, for 5 min. in eachbath.

Step 5.

Impregnation is completed by incubation in four baths of molten paraffinplaced within a large dessicator resting in a glycerin bath at about 75°C. to 80° C. and a reduced pressure of about 640 mm of Hg, for 5 mineach. Tissue sections were transferred from one paraffin bath to thenext at 5 min intervals, for a total impregnation time of 20 min. Each 5min interval was measured for the time that the pressure reading isabout 640 mm of Hg.

Example 4

Detection of Antigen in Tissue Sections

Paraffin sections are cut on a microtome to a thickness of 3 microns,placed in a water bath, and floated onto a glass slide. Paraffin wasmelted by placing slides in either a 58° C. oven for 30 minutes, orpreferably in a 37° C. oven for approximately 18 hours or overnight, andthen dewaxed in a xylene bath for 10 minutes. Slides were rehydrated indecreasing ethanol solutions for 1 min each (two baths of absolute, twobaths of 95%, and one bath of 90%) and rinsed by submerging in tap waterfor 2 min.

Endogenous peroxidase was blocked with a solution of 6% hydrogenperoxide (H₂O₂) and methanol, or 35 ml of 6% H₂O₂ with 140 ml methanol,incubated for 15 min. Slides were rinsed by submerging in tap water for2 min and PBS for 2 min, then dried.

Slides were transferred to a humidity chamber and normal horse serum(NHS) was added to block for 10 min. Excess normal horse serum wasdecanted from slides, and specific primary antibody was incubated for 30min on the tissue section in a humidity chamber at room temperature.Slides were flushed with PBS with back and forth motion using a squeezebottle, submerged in a PBS bath for 2 min, and excess PBS was dried offeach slide. Linking solution (also known as secondary antibody orbiotinylated anti-rabbit or anti-mouse) was added to each tissue sectionand incubated for 25 min in a humidity chamber. Slides were flushed withPBS using a squeeze bottle, submerged in a PBS bath for 2 min, andexcess PBS was dried off each slide.

Signal was developed according to manufacturer's instructions (VectorLaboratories). ABC solution was added to the tissue section andincubated for 25 min in humidity chamber. Slides were flushed with PBSin a squeeze bottle and submerged in a rack in a PBS bath for 2 min. Therack was submerged in a bath of DAB chromogen for 6 min, then submergedunder running water to wash gently for 4 min. Tissue sections werecounterstained with hematoxylin (staining time will depend on the age ofthe hematoxylin) from 15 sec to 90 sec. Slides were washed under runningwater for 3 min to remove excess counterstain, dehydrated in alcoholbaths (about 10 sec in each) from 85% to 100%, cleaned in xylene, andcoverslipped.

Better antigen reactivity has been shown for progesterone receptor,factor VIII-related antigen, CD-31, CD-68, cytokeratin-7, chromogranin,and smooth muscle antigen, probably because of better preservation ofantigen.

Reagents Catalog # Source Microscope slides - snow 00206 Surgipath coatX-TRA Elite ABC Kit (standard) PK-6100 Vector Laboratories Biotinylatedanti-mouse BA-2000 Vector Laboratories IgG (H & L) Biotinylatedanti-mouse BA-2020 Vector Laboratories IgM (H & L) Biotinylatedanti-mouse/anti-rabbit BA-6000 Vector Laboratories IgG (H & L) Normalhorse serum (NHS) S-2000 Vector Laboratories Diaminobenzidine K3466 DAKOCorporation tetrahydrochloride Potassium phosphate (monobasic) 7100-500NY Baxter Scientific Sodium phosphate (dibasic) 7917-2.5 NY BaxterScientific Sodium chloride (AR Crystals) 7581-2.5 NY Baxter Scientific30% Hydrogen peroxide 5240-500 NY Baxter Scientific Xylene 8644-20 NYBaxter Scientific Harris hematoxylin S-7735-3 Baxter Scientific Methylalcohol 3016-20 NY Baxter Scientific 95% Alcohol Florida DistillersAbsolute Ethyl Alcohol Florida Distillers

Antibodies, Dilutions and Incubation Times

Rabbit (R) Microwave (M) 30′ Incubation Mouse (MigG) Trypsin (T) 45′Incubation Mouse (MigM) Protease (P) 90′ Incubation Goat (G) Fast Green(FG)

Special Incubation Linking Abbreviation Antibody Procedure Time Solution(ACTH) Adrenocorticotropin 1:2000 30′ R Hormone (AACT) Alpha-1 1:5000030′ R Antichymotrypsin (AAT) Alpha-1 Antitrypsin 1:2000 30′ R (ADENO)Adenoviurs 1:1000 30′ MIgG (AFP) Alpha Fetoprotein 1:2500 30′ R (AEI/3)Cytokeratin 1:200 (M) 45′ MIgG (ALA) Alpha Lactalbumin 1:600 30′ R(ACTIN) Actin Muscle 1:200 30′ MIgG (APP-A4) Anti-Alzheimer 1:500 (M)45′ MIgG Precursor Protein A4 (ASPE) Aspergillus 1:500 30′ R (AR)Androgen Receptor 1:20 (M) (FG) 45′ MIgG (BCA) B-Cell 1:200 30′ MIgG(bcl-2) Anti-Human 1:100 (M) 45′ MIgG Oncoprotein (BerEp4) HumanEpithelial 1:25 30′ MIgG Antigen (B72.3) TAG72 1:100 30′ MIgGTumor-Associated Glycoprotein 72 (BLA36) B Lymphocyte 1:100 30′ MIgGAntigen (CMV) Cytomegalovirus 1:50 (P) 30′ MIgG (CHRG) Chromogranin 1:5030′ MIgG (CALC) Calcitonin 1:2000 30′ R (CEA) Carcinoembryonic 1:600030′ R Antigen (CERb′B2) c-erbB-2 Oncogene 1:1500 90′ R Mab1

Antibodies, Dilutions and Incubation Times

Special Incubation Linking Abbreviation Antibody Procedure Time Solution(CATH) Cathepsin D 1:2000 (M) 45′ R (CAM 5.2) Cytokeratin 1:500 (M) 45′R (CK 7) Cytokeratin 1:200 (M) 45′ MIgG (CK 20) Cytokeratin 1:25 (M) 45′MIgG (COLL IV) Collagen IV 1:25 (P) 30′ MIgG (CA 125) Anti-Human 1:20(M) 45′ MIgG CA 125 (MII) (CD 30) Anti-Human 1:200 (M) 45′ MIgG Ki-1Antigen (BER-H2) (ER) Estrogen Receptor 1:50 (M) (FG) 45′ MIgM (FVIII)Von Willebrand 1:50 (P) 30′ MIgM Factor (FSH) Follicle Stimulating1:3000 30′ R Hormone (5 HT) Serotonin 1:50 30′ MIgM (FXIII)Anti-coagulation 1:1200 30′ R Factor (GAST) Gastrin 1:2000 30′ MIgM(GFAP) Glial Fibrillary 1:1500 30′ R Acidic Protein (GLUC) Glucagon1:10000 30′ R (GH) Growth Hormone 1:5000 30′ R (GCDFP) Gross Cystic1:250 30′ MIgM Disease Fluid Protein (GRP) Gastrin-Releasing 1:1000 30′R Peptide (HMWK) High Molecular 1:10 45′ MIgM Weight Keratin (34 βE12)(Hbcore) Hepatitis B 1:5000 30′ R Core Antigen (HBsAg) Hepatitis B 1:10030′ MIgM Surface Antigen (HSV I) Herpes Simplex 1:10 30′ R Type I (HSVII) Herpes Simplex 1:10 30′ R Type II (HCG) Human Chorionic 1:50000 30′R Gonadotropin (HPL) Human Placental 1:100000 30′ R Lactogen

Antibodies, Dilutions and Incubation Times

Special Incubation Linking Abbreviation Antibody Procedure Time Solution(HIST) Histoplasma 1:1000 30′ R (H.Pyl) Heliobacter pylori 1:500 (M) 45′R (β-HCG) β-Human 1:10000 30′ R Chorionic Gonadotropin (IgA) Alpha Heavy1:400 30′ R Chain (IgG) Gamma Heavy 1:1000 30′ R Chain (IgAs) SecretoryPiece 1:200 30′ R of IgA (IgM) Mu Heavy 1:1000 30′ R Chain IgM (INS)Insulin 1:100 30′ R (Ki-67) Nuclear Antigen 1:50 (M) (FG) 45′ MIgG MIB-1(K) Kappa Light Chain 1:200 (M) 45′ MIgG (KERATIN) AEI/3 CAM 1:50/1:500(M) 45′ MIgG (LCA) Leucocyte Common 1:50 30′ MIgG Antigen (Leu M1) LeuM1 Antigen 1:200 (M) 45′ MIgM (Leu 7) Leu 7 Antigen 1:50 (M) 45′ MIgM(Lectin) Lectin 1:4000 USE INSTEAD OF NHS (Anti-Lectin) Anti-LectinAntigen 1:10000 30′ G (LEA 135) Anti-Human 1:50 30′ MIgG LuminalEpithelial Antigen (LH) Luteinizing 1:3000 30′ R Hormone (L) LambdaLight Chain 1:6000 (M) 45′ MIgG (LMK-8) Low Molecular 1:25 (M) 45′ MIgGWeight Keratin (LIP-AS Lipase 1:400 30′ MIgG 105) (MCA) MyeloidHistiocyte 1:400 (M) 45′ MIgG Antigen (MAC 387) (MUR) Muramidase 1:200030′ R (MYOGL) Myoglobin 1:5000 30′ R (MAPH) Macrophage 1:50 30′ MIgG

Antibodies, Dilutions and Incubation Times

Incuba- Special tion Linking Abbreviation Antibody Procedure TimeSolution (MTLT) Metallothionein 1:50 30′ MIgG (MEL) Melanoma HMB 45 1:5030′ MIgG (MAK 6) Anti-Cytokeratin 1:50 (T) 90′ MIgG (MBP) Myelin 1:50030′ R (MESO) Mesothelial Antigen 1:500 30′ MIgM (MAST-C) Mast Cell1:2000 (T) 30′ MIgG (MPO) Myeloperoxidase 1:5000 30′ R (MGN) Myogenin1:15 45′ MIgG (NB) Neuroblastoma 1:200 90′ MigG (N-FIL) N-Filament(2F11) 1:250 30′ MigG (NSE) Neuron Specific 1:4000 (M) 45′ MigG Enolase(PAMYL) Pancreatic Amylase 1:20 30′ MigG (PCP) Pneumocystis carinii 1:2530′ MigM (PLAP) Placental Alkaline 1:800 30′ R Phosphatase (PPP)Pancreatic Polypeptide 1:3000 30′ R (PTH) Parathyroid Hormone 1:250 (M)45′ (RAT) (PROL) Prolactin 1:500 30′ R (PAPH) Prostatic Acid 1:4000 30′R Phosphatase (PML) Progressive Multifocal 1:10000 30′ R (SV4O)Leucoencephalopathy (PR) Progesterone Receptor 1:100 (M) 45′ R (PR 1A6)Progesterone Receptor 1:50 (M) 45′ MigG (PSA) Prostate Specific 1:75030′ R Antigen (PCNA) Proliferating Cell 1:100 (M) (FG) 45′ MigG Nuclear(PS2) PS2 Protein 1:1000 45′ R (P53) p53 Antigen 1:50 (M) (FG) 45′ MigG

Antibodies, Dilutions and Incubation Times

Special Incubation Linking Abbreviation Antibody Procedure Time Solution(S100 A) S100 A Protein 1:3000 30′ R (S100) S100 Protein 1:2000 30′ R(SOMAT) Somatostatin 1:3000 30′ R (SYNAP) Synaptophysin 1:800 (M) 45′ R(SMA) Smooth Muscle 1:100 30′ MigG Actin (∝SR-1) Sarcomeric Actin 1:10030′ MigG (TESTOS) Testosterone 1:250 30′ R (TGB) Thyroglobulin 1:2000030′ R (TP-103) Treponema 1:50 (T) 30′ MigG (TM) Thrombomodulin 1:50 30′MigG (TSH) Thyroid Stimulating 1:2000 30′ R Hormone (TCA) T-Cell Antigen1:800 (M) 45′ MigG (TOXO) Toxoplasma 1:1000 30′ R (UBT) Ubiquitin 1:25030′ R (VIP) Vasoactive intestinal 1:1500 30′ R peptide (VIM) Vimentin1:800 (M) 45′ MigG (VZV) Variecella-Zoster 1:100 30′ MigG Virus (WSKER)Wide Spectrum 1:500 30′ R Keratin

Example 5

DNA Extraction from Processed Tissue Sections

Two six micron tissue sections were placed in a 1.5 ml microfuge tube,800 μl xylene was added and mixed by vortexing, 400 μl absolute ethanolwas added and mixed by vortexing, the tube was centrifuged for 5 minutesin a high speed microfuge, and the supernatant was decanted. To thepellet, 800 μl absolute ethanol was added and mixed by vortexing.

The supernatant was decanted after centrifugation as above, and 100 μlof a detergent/proteinase K solution (1% NP40 or Triton X-100, 2.4 μl of2.5 mg/ml proteinase K) was added to the pellet and incubated at 55° forone hour. Proteinase K was inactivated by incubation at 95° for 10 min.Save the supernatant containing DNA after centrifugation in themicrofuge for 5 min. This material is ready for PCR. It should beprecipitated and/or extracted further if Southern blotting is planned.More sections would be required to obtain enough DNA for restrictionanalysis.

FIG. 13A shows the quantity and quality of polymerase chain reaction(PCR) amplified DNA are comparable between samples prepared according tothe present invention (Example 1) and by conventional tissue processing(Tissue-Tek VIP histoprocessor, Miles-Sakura, used according tomanufacturer's instructions).

Example 6

RNA Extraction from Processed Tissue Sections

Ten sections (7 μm each) of a paraffin block were cut using disposableblades; the blocks were prepared according to the present invention andby conventional tissue processing as described in Example 5. They wereplaced in 50 ml Falcon tubes, deparaffinized with 20 ml of xylene, andthe remaining tissue was then washed twice with absolute alcohol for 30minutes. The tissue was suspended at 0.5 g/ml in a solution containing4M guanidinium thiocyanate, 25 mM Na citrate pH 7.0, 0.5%N-laurylsarcosine, and 0.1 M of 2-mercaptoethanol. The solution wasmixed by vortexing and DNA was sheared by passage through an 18 to 22gauge syringe needle.

The RNA-containing solution was carefully layered on 2.8 ml of 5.7 MCsCl in several 5 ml centrifuge tubes (Sorvall), and RNA was sedimentedby centrifugation in an SW55Ti rotor at 35,000 rpm and 18° C. for 14hours in a Beckman L8-53 ultracentrifuge. The top fraction was carefullyremoved to leave an RNA pellet at the bottom of the tube. The pellet wasresuspended with ribonuclease-free water, and the Eppendorf tube wasspun at 14,000 rpm for 10 min. The supernatant containing RNA was savedand the UV absorbance was measured: extinction coefficient 1 OD₂₈₀/cm is40 μg/ml RNA, OD₂₆₀/OD₂₈₀ ratio should be between about 1.8 and about2.0. A total of 45 μg RNA was extracted from tissue specimens preparedaccording to the present invention whereas no RNA was detectable fromtissue specimens processed conventionally (FIG. 13B).

While the present invention has been described in connection with whatis presently considered to be practical and preferred embodiments, it isunderstood that the present invention is not to be limited or restrictedto the disclosed embodiments but, on the contrary, is intended to covervarious modifications and equivalent arrangements included within thespirit and scope of the appended claims.

Thus, it is to be understood that variations in the described inventionwill be obvious to those skilled in the art without departing from thenovel aspects of the present invention and such variations are intendedto come within the scope of the claims below.

We claim:
 1. A method of processing a tissue specimen for histologycomprising: (a) slicing a solid tissue to provide at least one tissuespecimen to a thickness of up to 2 mm, (b) fixing the tissue specimen,(c) dehydrating the tissue specimen, (d) removing fat from the tissuespecimen, (e) impregnating the tissue specimen with a mixture of waxeshaving different melting points to provide a block comprised of thetissue specimen and wax, and (f) sectioning the block to provide atissue section which is only then placed on a slide; wherein said tissuespecimen is processed in steps (b)-(d) by a series of all non-aqueoussolutions, at least one of which is a first solution comprising fixativeand dehydrating agent; wherein fixation, dehydration, and fat removaloccur with agitation of said non-aqueous solutions and heating bymicrowave energy; wherein impregnation is conducted under less thanatmospheric pressure; and whereby said tissue specimen is fixed,dehydrated, defatted, and impregnated in about 90 minutes or less.
 2. Amethod of continuous tissue processing for histology comprising: (a)providing a series of tissue specimens up to 1.5 mm in thickness, (b)fixing at least one of said tissue specimens, (c) dehydrating at leastone of said tissue specimens, (d) removing fat from at least one of saidtissue specimens, and (e) impregnating at least one of said tissuespecimens under less than atmospheric pressure with a mixture of waxeshaving different melting points, wherein steps (b)-(e) are completed inabout 90 minutes or less; wherein all solutions for processing saidtissue specimens are non-aqueous and said series of tissue specimens isprocessed by initiating step (b) for a later-processed tissue specimenbefore step (e) for an earlier-processed tissue specimen has beencompleted, and wherein steps (b), (c), and (d) occur with agitation andheating by microwave energy.
 3. A method of continuous tissue processingfor histology comprising: (a) slicing a solid tissue to provide a tissuespecimen of up to 2 mm in thickness, (b) fixing and dehydrating saidtissue specimen by incubation in a series of all non-aqueous solutionswith agitation and heating by microwave energy, (c) impregnating saidfixed and dehydrated tissue specimen under less than atmosphericpressure, and (d) repeating steps (b) and (c) at least once with anothertissue specimen, wherein at least one repetition of steps (b) and (c)takes about 90 minutes or less; whereby tissue specimens arecontinuously processed by initiating step (b) for a later-processedtissue specimen before step (c) has been completed for anearlier-processed tissue specimen.
 4. A method according to claim 3,wherein said tissue specimen is sliced to a thickness between 1 mm and 2mm before processing.
 5. A method according to claim 3, wherein saidtissue specimen is sliced to a thickness between 1 mm and 1.5 mm beforeprocessing.
 6. A method according to claim 3 further comprisingsectioning said fixed, dehydrated, and impregnated tissue specimen toprovide a tissue section of between 1 micron and 50 microns.
 7. Themethod of claim 3, wherein at least one of said series of non-aqueoussolutions is a first solution comprised of fixative and dehydratingagent.
 8. The method of claim 7, wherein said tissue specimen isincubated in said first solution for at least 15 minutes.
 9. The methodof claim 7, wherein said first solution is comprised of alcohol andketone.
 10. The method of claim 9, wherein said first solution containsa volume ratio of alcohol to ketone between 1:1 and 3:1.
 11. The methodof claim 7, wherein said first solution is further comprised of polymersbetween 100 and 500 average molecular weight.
 12. The method of claim11, wherein said first solution is further comprised of a surfactant atabout 1% of total volume.
 13. The method of claim 3, wherein at leastone of said series of non-aqueous solutions is a first solutioncomprised of acetone, isopropyl alcohol, and low molecular weightpolyethylene glycol.
 14. The method of claim 3 further comprisingclearing said tissue specimen in a non-aqueous solution comprised ofclearant.
 15. The method of claim 14, wherein said clearant is xylene.16. The method of claim 3, wherein tissue specimens are processedwithout using formalin.
 17. The method of claim 3, wherein tissuespecimens are processed without using xylene.
 18. The method of claim 3,wherein at least one of said series of non-aqueous solutions iscomprised of alcohol, ketone, and mineral oil.
 19. The method of claim3, wherein said fixed and dehydrated tissue specimen is impregnated witha mixture of waxes having different melting points.
 20. The method ofclaim 3, wherein said fixed and dehydrated tissue specimen isimpregnated with a wax solution comprised of xylene and paraffin. 21.The method of claim 3, wherein said fixed and dehydrated tissue specimenis impregnated with a wax solution comprised of mineral oil andparaffin.
 22. The method of claim 3, wherein said fixed and dehydratedtissue specimen is impregnated in molten paraffin for at least 12minutes.
 23. A method of continuous tissue processing for histologycomprising: (a) slicing a solid tissue to provide tissue specimens withthickness between 1 mm and 2 mm, wherein a tissue specimen to beprocessed is not placed on a slide for histology until said tissuespecimen has been hardened, impregnated, and sectioned; (b) hardeningsaid tissue specimen by incubation in a series of all non-aqueoussolutions with agitation and heating by microwave energy, whereinincubation in a first non-aqueous solution comprised of ketone andalcohol is then followed by incubation in a second non-aqueous solutioncomprised of less concentrated ketone and more concentrated alcohol ascompared to said first non-aqueous solution; (c) impregnating saidhardened tissue specimen in a wax solution under less than atmosphericpressure to provide a block comprised of said hardened and impregnatedtissue specimen and wax, wherein hardening and impregnating a tissuespecimen takes about 90 minutes or less; and (d) repeating steps (b) and(c) at least once with another tissue specimen; whereby tissue specimensare continuously processed by initiating hardening of a later-processedtissue specimen before impregnation of an earlier-processed tissuespecimen has been completed.
 24. A method according to claim 23 furthercomprising sectioning said hardened and impregnated tissue specimen toprovide a tissue section of between 1 micron and 50 microns.
 25. Themethod of claim 23 further comprising placing said tissue section on aslide for histology, and then removing wax from the slide.
 26. Themethod of claim 23, wherein said tissue specimen is incubated in saidfirst non-aqueous solution for at least 15 minutes.
 27. The method ofclaim 23, wherein said first non-aqueous solution is comprised ofacetone and isopropyl alcohol.
 28. The method of claim 27, wherein saidfirst non-aqueous solution contains a volume ratio of alcohol to ketonebetween 1:1 and 3:1.
 29. The method of claim 23, wherein said firstnon-aqueous solution is further comprised of polymers between 100 and500 average molecular weight.
 30. The method of claim 29, wherein saidfirst non-aqueous solution is further comprised of a surfactant at about1% of total volume.
 31. The method of claim 23, wherein said firstnon-aqueous solution is comprised of acetone, isopropyl alcohol, and lowmolecular weight polyethylene glycol.
 32. The method of claim 23 furthercomprising clearing said tissue specimen in a non-aqueous solutioncomprised of clearant.
 33. The method of claim 32, wherein said clearantis xylene.
 34. The method of claim 23, wherein tissue specimens areprocessed without using formalin.
 35. The method of claim 23, whereintissue specimens are processed without using xylene.
 36. The method ofclaim 23, wherein at least one of said series of non-aqueous solutionsis comprised of alcohol, ketone, and mineral oil.
 37. The method ofclaim 23, wherein said fixed and dehydrated tissue specimen isimpregnated with a mixture of waxes having different melting points. 38.The method of claim 23, wherein said fixed and dehydrated tissuespecimen is initially impregnated with a wax solution comprised ofxylene and paraffin.
 39. The method of claim 23, wherein said fixed anddehydrated tissue specimen is impregnated with a wax solution comprisedof mineral oil and paraffin.
 40. The method of claim 23, wherein saidfixed and dehydrated tissue specimen is impregnated in molten paraffinfor at least 12 minutes.